Chromosomes are separated into regions of distinct transcriptional activity. Key to the establishment of these regions is the presence of specific histone post-translational modifications (PTMs) on chromatin. These epigenetic marks provide signals that recruit proteins to activate or repress transcription. The precise marking of a given histone (or particular combination of markings) is key for driving the cellular program of gene expression. The mis-regulation of histone PTM addition or removal has been linked to diseases such as cancer. Current technologies used to study histone PTMs are largely limited by the analysis of a single histone PTM in context of chromosomal location, while it appears that the combinatorial nature of histone PTM addition is key for transcriptional regulation. We outline the development of a novel technology that will enable epigeneticists to isolate a given region of a chromosome (on the order of 5 nucleosomes), identify the contained and co-occupancy of histone PTMs, identify the proteome component of the particular region of chromatin and control for non-specifically interacting proteins/PTMs. This procedure will be referred to as ChAP-MS for chromatin affinity purification with mass spectrometry. The working hypothesis of our proposed work is: development of the ChAP-MS technology to specifically enrich small chromosome fragments for mass spectrometric analysis will enable epigeneticists to perform whole epigenome and chromosome proteome studies for the first time. Our objectives are 1) Development of a chromatin affinity purification (ChAP) technology that will permit efficient isolation of specific chromosomal fragments for use in epigenomic and proteomic profiling, 2) Devise an unambiguous readout strategy to be used in conjunction with ChAP analysis that permits discrimination between specific and non-specific protein/histone interactions with a defined chromosomal fragment, 3) Design microscale protocols to be used in conjunction with ChAP analysis for separating each core histone component of a given chromosomal fragment on the basis of PTM occupancy, thus allowing mass spectrometric profiling of histone PTMs, and 4) Use ChAP-MS to profile the epigenome and proteome of a histone acetyltransferase complex. PUBLIC HEALTH RELEVANCE: Understanding how histones are post-translationally modified will enable researchers to better understand the molecular mechanism of diseases such as cancer. Current technologies lack the ability to thoroughly analyze the combinatorial nature of these modifications in context of chromosomal location. We plan to develop a new technology that will provide a manner to specifically analyze these combinatorial modifications in the chromosomal context, thereby providing a new avenue for researchers to understand complex diseases.